Spectrally resolved infrared microscopy and chemometric tools to reveal the interaction between blue light (470 nm) and methicillin-resistant Staphylococcus aureus

https://doi.org/10.1016/j.jphotobiol.2016.12.030Get rights and content

Highlights

  • FTIR measurements and chemometric tools to evaluate the photoreactivity of MRSA

  • Uncovering the mechanism underlying the bactericidal effect of blue light (470 nm)

  • UV (253.5 nm) and blue light (470 nm) use different stratagems to modify DNA.

  • Irradiation of MRSA with blue light (470 nm) induces A-DNA cleavage.

  • Colony Forming Units (CFU) Assay

Abstract

Blue light inactivates methicillin-resistant Staphylococcus aureus (MRSA), a Gram-positive antibiotic resistant bacterium that leads to fatal infections; however, the mechanism of bacterial death remains unclear. In this paper, to uncover the mechanism underlying the bactericidal effect of blue light, a combination of Fourier transform infrared (FTIR) spectroscopy and chemometric tools is employed to detect the photoreactivity of MRSA and its distinctive pathway toward apoptosis after treatment. The mechanism of action of UV light and vancomycin against MRSA is also investigated to support the findings. Principal component analysis followed by linear discriminant analysis (PCA- LDA) is employed to reveal clustering of five groups of MRSA samples, namely untreated (control I), untreated and incubated at ambient air (control II), irradiated with 470 nm blue light, irradiated with 253.5 UV light, and vancomycin-treated MRSA. Loadings plot from PCA-LDA analysis reveals important functional groups in proteins (1683, 1656, 1596, 1542 cm 1), lipids (1743, 1409 cm 1), and nucleic acids region of the spectrum (1060, 1087 cm 1) that are responsible for the classification of blue light irradiated spectra and control spectra. Cluster vector plots and scores plot reveals that UV light-irradiated spectra are the most biochemically similar to blue light- irradiated spectra; however, some wavenumbers experience a shift. The shifts between blue light and UV light irradiated loadings plot at νasym PO2  band (from 1228 to 1238 cm 1), DNA backbone (from 970 to 966 cm 1) and base pairing vibration of DNA (from 1717 to 1712 cm 1) suggest distinctive changes in DNA conformation in response to irradiation. Our findings indicate that irradiation of MRSA with 470 nm light induces A-DNA cleavage and that B-DNA is more resistant to damage by blue light. Blue light and UV light treatment of MRSA are complementary and distinct from the known antimicrobial effect of vancomycin. Moreover, it is known that UV-induced cleavage of DNA predominantly targets B-DNA, which is in agreement with the FTIR findings. Overall the results suggest that the combination of light and vancomycin could be a more robust approach in treating MRSA infections

Introduction

Methicillin-resistant Staphylococcus aureus (MRSA) is a Gram-positive bacterium, which infects the skin, soft tissues. Infection generally begins as swollen painful red bumps, but can quickly turn into deep, painful abscesses that require surgical draining. Infection can become life threatening when it involves deep tissues, such as bones, joints, surgical wounds, the bloodstream, heart valves and lungs. To combat MRSA infection, a myriad of antibiotics has been used; however, their effectiveness against staphylococci infections has decreased substantially due to the evolution of resistant strains of bacteria [1], [2]. The search for more efficacious remedies and alternative measures to address the problem has intensified. Therapies under investigation include many natural and synthetic products such as antibacterial clay [3], combined honey and antibiotics [4], hyperbaric oxygen [5], photodynamic therapy [6], [7], [8], porphyrine [9] and tetraaryl-porphyrin photosensitizers [10], blue light phototherapy [11], [12], [13], 207 nm UV light therapy [14] and combined blue light and hyperbaric oxygen therapy [15].

Photo-irradiation to inactivate MRSA in vitro is gaining interest. Previously two strains of MRSA are successfully eradicated with 405 nm and 470 nm light in vitro, and more recently, 405 nm, 470 nm and 415 nm blue light have been shown to inactivate cultures of Staphylococcus aureus (both MRSA and methicillin susceptible Staphylococcus aureus (MSSA)), Escherichia coli and other bacterial pathogens [16], [17]; however, the mechanism of bacterial cell death remains unclear. Hence, the focus of this study is to determine blue light induced cellular and intracellular biochemical changes in MRSA. Blue light induced cell death is compared with cell death induced by UV-irradiation, which is known to initiate DNA cleavage. The major finding in this paper indicates that blue light irradiation on MRSA is a complementary or alternative treatment to the existing techniques, and elicits a similar response as UV light, but targets different DNA conformation.

Fourier transform infrared (FTIR) spectroscopy, a nondestructive technique, provides information on quantitative profile of overall biochemical composition of cells and tissues. Recently, the biological and clinical applications of FTIR spectroscopy, driven by advanced computational analysis have been developed [18], [19], [20], [21]. Identification and discrimination of microbial species and subspecies by FTIR spectroscopy has been shown in several studies [22], [23], [24], [25], [26], [27]. The FTIR spectrum of MRSA shows important bands attributed to proteins, lipids and nucleic acids in the biochemical spectral region 1800–900 cm 1. Other spectroscopic methods including raman spectroscopy [28], [29], [30] and mass spectrometry [31] have also been applied to monitoring and identifying S. aureus species.

Here we present, for the first time, the mechanism of action underlying the blue light inactivation of MRSA by FTIR spectroscopy coupled with principal component analysis followed by linear discriminant analysis (PCA-LDA). Moreover, in this study, the mode of action of UV light and vancomycin against MRSA is investigated. Spectral data were loaded into PCA algorithm to reduce the dimensionality of the data before employing LDA algorithm to reveal clustering [32]. This method enables us to extract important spectral biomarkers differentiating treated and control MRSA. Infrared bands attributed to different conformations of DNA are seen in blue light and UV light irradiated MRSA, when compared to control MRSA. The vancomycin-treated MRSA is the most distinct, and is dominated by the vancomycin absorption bands due to high dosage (40 μg/mL) of the antibiotic to maximally suppress MRSA growth.

Section snippets

Materials and Methods

Five different experimental groups of samples are evaluated using FTIR. Group I: Control I, comprised of untreated MRSA incubated at 37 °C for 24 h (N = 3); Group II: Control II, comprised of untreated MRSA incubated in ambient air for 24 h (N = 3); Group III: Irradiation with 262 J/cm2 of blue 470 nm light, incubated at 37 °C for 24 h (N = 5); Group IV: UV light- irradiated (253.5 nm) MRSA, incubated at 37 °C for 24 h (N = 5); Group V: Vancomycin-treated MRSA, incubated at 37 °C for 24 h (N = 3). The rationale for

Results and Discussion

Colony-forming units (CFU) are determined on control and treated MRSA (collected posterior to FTIR measurements) to characterize the effect of treatment or biological influence on bacteria or colony formation. The results for the two control groups are indicative of functioning MRSA. The light treated groups do not express any bacterial growth while vancomycin-treated bacteria still demonstrates negligible colonies of bacteria as anticipated (Fig. 1). Thus since radiant exposure cumulative

Acknowledgments

Financial support was received from the Stimulus Program to Accelerate Research Clusters (SPARC) of College of Health Sciences, University of Wisconsin-Milwaukee (Grant No. PRJ73CL to J.E and V.V.B), NSF grants CHE-1508240 and CHE-1112433 to C.J.H.

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